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. 2014 Nov;96(5):931-8.
doi: 10.1189/jlb.4A1113-604RR. Epub 2014 Jul 31.

V体育平台登录 - LL-37 modulates human neutrophil responses to influenza A virus

Shweta Tripathi  1 , Anamika Verma  1 , Eun-Jeong Kim  1 , Mitchell R White  1 , Kevan L Hartshorn  2
Affiliations

LL-37 modulates human neutrophil responses to influenza A virus

Shweta Tripathi et al. J Leukoc Biol. 2014 Nov.

"V体育官网入口" Abstract

Recent studies have shown that the human cathelicidin, LL-37, has antiviral activity against IAV in vitro and in vivo. Neutrophils are important cellular components of the initial innate response to IAV infection. In addition to its direct antimicrobial activities, LL-37 has important immunomodulatory effects. In this study, we explore how LL-37 affects interactions of IAV with human neutrophils. LL-37 did not alter neutrophil uptake of IAV but significantly increased neutrophil H2O2 responses to the virus. IAV stimulated production of NETs in vitro, and this response was increased by preincubating the virus with LL-37 VSports手机版. NADPH-oxidase blockade did not reduce IAV-induced NET formation or the increased NET response stimulated by LL-37 + IAV. The increased respiratory burst and NET responses were, however, inhibited by preincubating cells with a formyl peptide receptor blocker, indicating that LL-37 engages these receptors when complexed with IAV. Responses to IAV alone were not inhibited by formyl peptide receptor blockade. It has been reported that LL-37 reduces proinflammatory cytokine responses during IAV infection in vivo. We now show that IAV alone potentiated release of IL-8 from neutrophils, and preincubation with LL-37 reduced IAV-stimulated IL-8 release. These results confirm that LL-37 modulates human neutrophil responses to IAV in a distinctive manner and could have important bearing on the protective effects of LL-37 during IAV infection in vivo. .

Keywords: FPRL-1; antimicrobial peptides; defensin; innate immunity V体育安卓版. .

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Figures

Figure 1.
Figure 1.. Effects of LL-37 on neutrophil uptake of IAV.
The virus was first preincubated with LL-37 or HNP-2 for 30 min at 37°C, and aliquots of these samples were then added to the neutrophils. The concentrations of LL-37 shown are those present in the final incubation with neutrophils, and they are given as molar concentrations to allow comparison of the effective concentrations of HNP2. The multiplicity of infection (MOI) for these experiments was ∼12 (or 12 infectious virions/neutrophil). HNP-2 caused significant increases in viral uptake, but LL-37 did not. Results are mean ± sem of five experiments using separate blood donors. *P < 0.05 compared with control using Student's t-test.
Figure 2.
Figure 2.. Neutrophil H2O2 production in response to IAV, LL-37, or IAV + LL-37.
Neutrophils were treated with control buffer, IAV, LL-37, or combinations of IAV and LL-37, and H2O2 responses were measured by the fluorescent scopoletin assay. Decrease in scopoletin fluorescence corresponds to H2O2 production. (A) Results of treating neutrophils with IAV alone or combinations of IAV and LL-37. In these experiments, the virus and LL-37 were preincubated for 30 min at 37°C before addition to the neutrophils. These samples were diluted eightfold upon addition to the neutrophils (e.g., the sample noted as 4 μg/ml LL-37 was originally 32 μg/ml during the initial incubation with IAV). The concentrations of LL-37 shown are those present in the final incubation with neutrophils. The MOI for these experiments was 40. IAV alone elicited increased H2O2 generation compared with control buffer. Preincubation of IAV with LL-37 caused significant further increases in H2O2 generation in a dose-related manner compared with IAV alone. Doses of LL-37 ≥ 0.75 μg/ml caused significantly greater H2O2 generation than IAV alone (P<0.05). (B) Effects of preincubating neutrophils with LL-37, followed by adding IAV. In these experiments, LL-37 was added to the neutrophils first, and then, virus was added without washing off the LL-37. In this case, only the highest concentration of LL-37 (4 μg/ml) caused an increase in H2O2 generation compared with IAV alone (P<0.05 compared with IAV alone). For comparison, the results with LL-37 (4 μg/ml) alone (i.e., with no virus) are shown. The results shown are mean ± sem of nine experiments using separate neutrophil donors for each experiment. *Significant increase in H2O2 generation (P<0.05) compared with IAV alone, as measured by Student's t-test.
Figure 3.
Figure 3.. Effect of FPRL-1 blockade on H2O2 generation in response to IAV or IAV + LL-37.
Experiments were performed as in Fig. 2. (A) Neutrophils were stimulated with IAV or the WKYMVM peptide alone or combined with the WRW4-blocking peptide. WRW4 markedly reduced the response to WKYMVM (P<0.001) but significantly increased the response to IAV (P<0.01). (B) IAV was pretreated with LL-37 (4 μg/ml), as in Fig. 2, and this again resulted in a significantly increased response compared with IAV alone (P<0.001). This response was reduced significantly by WRW4 (P<0.05), such that it was nearly the same at the response to IAV + WRW4 without LL-37. Results are mean ± sem of six experiments with separate neutrophil donors. *Significant increase in H2O2 generation compared with IAV alone (P<0.05) using Student's t-test; **WRW4 significantly reduced responses (P<0.05), as measured using Student's t-test.
Figure 4.
Figure 4.. Induction of NETs by IAV or PMA.
Neutrophils were exposed to control buffer, Phil82 IAV, or PMA (400 ng/ml) for 3 h, and Sytox Green fluorescence was measured by a plate-reading fluorometer (A) or microscopy (B). The MOI for these experiments was 50. Results in the graph are mean ± sem of six experiments, and separate neutrophil donors and microscopy results are representative of samples treated with control buffer, IAV, or PMA from these experiments. (B, left) Phase-contrast image to indicate how many neutrophils were present in a representative field. The three other panels were taken using fluorescent microscopy. The images were taken at 10× original magnification. *P < 0.05 compared with control buffer using Student's t-test.
Figure 5.
Figure 5.. Effects of LL-37 on neutrophil NET formation.
(A) Sytox Green fluorescence measurements of neutrophils treated with control buffer, PMA, IAV, IAV pretreated with the indicated concentrations (μg/ml) of LL-37, or LL-37 alone. In this set of experiments, the IAV samples preincubated with LL-37 were not diluted further upon addition to neutrophils. Black bars show results obtained with neutrophils preincubated with DPI, and DPI did not significantly reduce fluorescence of IAV- or IAV + LL-37-treated samples but did reduce that of samples treated with PMA. (B) Effects of WRW4 on NET formation in response to IAV, PMA, WKYMVM peptide, or IAV preincubated with LL-37. Results are mean ± sem of six experiments with separate neutrophil donors. (C) Fluorescent micrographs on NET formation in response to control buffer, IAV, IAV + LL-37 (32 μg/ml), or PMA. (Upper) Images show control neutrophils; (lower) neutrophils preincubated with DPI (10μM). Results are representative of three experiments with different neutrophil donors. The images were taken at 10× original magnification. *Fluorescence was increased significantly (P<0.01) compared with control cells alone, as measured by Student's t-test; **LL-37 caused significant increases (P<0.05) in NET response compared with IAV alone, as assessed by paired t-test; #LL-37 caused significant increases (P<0.05) in NET formation compared with IAV alone, as measured by ANOVA; &DPI or WRW4 significantly reduced (P<0.05) NET formation, as assessed by ANOVA.
Figure 6.
Figure 6.. Location of IAV with respect to NETs using confocal microscopy.
Confocal microscopic images from two representative experiments of neutrophils treated with control buffer, IAV, or IAV + LL-37 (32 μg/ml). DNA was stained blue with DAPI, cell membranes green with WGA-Oregon Green, and IAV red with Alexa Fluor 594. NET formation was present in many fields of IAV-exposed cells but was more evident in cells treated with IAV + LL-37. The images shown were taken at 100× original magnification.
Figure 7.
Figure 7.. Effects of IAV or IAV plus LL-37 on neutrophil IL-8 generation.
Neutrophils were stimulated with LPS, infectious IAV, or heat- or UV-inactivated IAV, and IL-8 production in the cell supernatants was measured 20 h later. LPS, infectious IAV, and UV-inactivated IAV all caused increased IL-8 production compared with control (untreated neutrophils). Before adding to neutrophils, the virus was preincubated with control buffer, LL-37, or sLL-37 for 30 min at 37°C. These samples were then further diluted 2.4-fold in addition to neutrophils. The amounts of LL-37 or sLL-37 were those present in the final incubation with neutrophils. The MOI for these experiments was 10. LL-37 reduced IL-8 generation in response to LPS or infectious IAV in a dose-related manner. sLL-37 did not reduce the IL-8 response to IAV. LL-37 or sLL-37 alone (in the absence of other stimuli) did not increase IL-8 generation compared with control. Results are mean ± sem of six experiments with separate neutrophil donors. *Significant increase at P < 0.05 compared with control, as measured by Student's t-test; **significant decreases in LPS induced IL-8 generation caused by LL-37, as assessed by Student's t-test; #significant decrease compared with IAV alone at P < 0.05, as measured by ANOVA.
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